The goal of this project is to identify novel chemotherapeutic targets for the treatment of diseases of mycobacterial origin. Initial work by this laboratory identified the gene for an enzyme which was sufficient to induce formation of the distal cis cyclopropane in the mycolic acids of Mycobacterium tuberculosis. This modification to a major cell-wall molcule was shown to have a role in contributing to the ability of the organism to withstand oxidative challenge, suggesting that this modification may play a role in macrophage survival. This gene sequence was used to identify, by homology, a second gene whose protein product was sufficient to induce formation of the proximal cyclopropane. This modification was shown to induce added structural stability to the outer membrane system. The abailability of these gene sequences allowed the isolation of a cluster of four genes which together act to form the methoxy mycolate series (the second most abundant constituent of the outer cell-wall matrix). By DNA sequence analysis and subcloning each gene separately and in all possible combinations, we assigned the function of three of the members of this gene cluster following heterologous expression in Mycobacterium smegmatis. These six enzymes were found to form a highly homologous family with at least 50% identity between the amino acid sequences of any two members. All members of this family possessed a highly conserved binding domain for S-adenosyl-L-methionine and the function of all known members can be interpreted as a variation on a methyl transfer reaction from SAM to an olefinic mycolic acid precursor. These studies have elucidated the biosynthetic pathway for methoxy mycolates and suggested that the structural variation in mycolic acids can be explained as a direct result of sequence variation within this family os SAM-dependent methyl transferases. The specific reaction products of each enzyme further suggest that such reactions may proceed through a common cationic intermediate. This implication has important ramifications for drug discovery since targeting such a common intermediate would allow the simultaneous targeting of the entire enzyme family making the development of drug resistance through a single mutational event highly unlikely.